1. Field of the Invention
The present invention relates to particles for a diagnostic agent. More particularly, the present invention relates to particles for a diagnostic agent, which are used for the measurement of biochemical substances, especially in medical tests, and to a turbidmetric immunoassay using the same.
2. Description of the Background
As an important means in clinical laboratory tests, a method is used in which an immune response substance, such as an antibody, an antigen, or the like, or a nucleic acid is provided on carrier particles, and the corresponding substance to be tested, such as antigen, antibody, enzyme, nucleic acid, or the like, is detected by a specific reaction.
For example, when a dispersion of polymer particles on which an antibody has been provided by physical adsorption or chemical bonding is prepared, and a liquid to be tested containing an antigen corresponding to the antibody is added to the dispersion, the polymer particles aggregate due to changes in the surface conditions of the particles or due to formation of crosslinking between particles caused by an antigen-antibody specific bond.
Although such an aggregation of polymer particles can be observed with the naked eye on an appropriate plate, the substance to be tested (antigen) in a liquid sample is determined generally by measuring changes in turbidity of the liquid sample caused by the aggregation.
Examples of such a measuring system (determination method) include a method in which the changing rate of turbidity after addition of a liquid sample is measured (rate assay) and a method in which the subequilibrium turbidity after a lapse of a predetermined time after addition of a liquid sample is measured (end point assay).
Each of these methods uses a calibration curve prepared in advance by measuring a relationship between the amount of the substance to be tested (concentration) and the turbidity (changing rate or semiequilibrium turbidity).
In such a calibration curve, it is necessary that the absorbance of the substance to be treated is increased monotonously without a pro-zone region from its low concentration to high concentration, independent of the regularity of the gradient.
Polystyrene particles or carboxylic acid-modified styrene particles are known as polymer particles constituting particles for a diagnostic agent. Examples of the carboxylic acid which is used in a small amount to obtain the carboxylic acid-modified styrene particles include methacrylic acid, acrylic acid, itaconic acid, fumaric acid, and the like.
These polystyrene particles (polystyrene particles or carboxylic acid-modified styrene particles) become particles constituting a diagnostic agent as a result that antibodies or the like are sensitized. Also, a physiological saline supplemented with a substances necessary for adjusting the diagnostic agent performance is used as the medium of the diagnostic agents.
However, when the turbidity generated by the aggregation of polymer particles is measured based on the absorbance, the known polystyrene latex causes a problem in that its absorbance exceeds the measuring limit of a test apparatus even within a low concentration range of a substance to be tested due to its large absorbance even under unaggrigated conditions (concentration of substance to be tested=0). Additionally, when the concentration of the polymer particles (latex concentration) is lowered to minimize the absorbance under unaggrigated conditions, a pro-zone phenomenon occurs at a high concentration region of the substance to be tested, so that considerable time and labor are required at the field of clinical laboratory tests, such as the necessity to carry out dilution of liquid samples.
In order to prevent generation of the pro-zone phenomenon in calibration curves by solving such problems, various resolving means, such as selection of antibodies etc., sensitization method, sensitization conditions, optimization of dispersion composition, and the like, have been examined, but satisfactory results have not been obtained.
Furthermore, as an approach from the material point of view for preventing generation of the pro-zone phenomenon in a calibration curve without exceeding the absorbance after aggregation over the measuring limit of a test apparatus, it has been proposed to use a fluorine-containing latex material which has a lower refraction index than that of the polystyrene latex (e.g., JP-A-61-247966, JP-A-63-200065, JP-A-63-278913 and JP-A-1-266111; the term xe2x80x9cJP-Axe2x80x9d as used herein means an xe2x80x9cunexamined published Japanese patent applicationxe2x80x9d).
However, the fluorine-containing latex material has a problem in terms of production cost, for example, due to the necessity to use a pressure vessel for its production. Also, since the latex particles have a large specific gravity of at least 1.5, the material is poor in dispersion stability and therefore causes another problem in terms of storage stability. Moreover, it has still another problem in view of the difficulty in redispersion after centrifugation due to its low glass transition temperature (Tg) and in immobilizing an antigen or antibody by physical adsorption.
On the other hand, the development of polymer particles stained or dyed with a dyestuff or fluorescent material has been improved in order to improve visibility of the test results and detection sensitivity and to carry out measurement and inspection of a biochemical substance using the color or fluorescence of the polymer particles as a marker.
However, nothing is known about polymer particles constituting the particles for a diagnostic agent having good staining and dyeing properties.
An object of the present invention is to provide a novel particle for a diagnostic agent, which comprises a specific polymer as its constituting component.
Another object of the present invention is to provide particles for a diagnostic agent, which, when the turbidity generated by the aggregation of particles is measured based on the absorbance, show a low absorbance under unaggrigated conditions (concentration of substance to be tested=0), so that the absorbance does not exceed measuring limit of a test apparatus within a broad concentration range and it does not generate a pro-zone phenomenon even at a high concentration region in measuring a calibration curve of the concentration of a substance to be tested and the absorbance.
Furthermore, an object of the present invention is to provide particles for a diagnostic agent, which can measure a calibration curve having a broad concentration range of a substance to be tested without causing a pro-zone phenomenon, even when the sensitization treatment is carried out under standard conditions.
Moreover, an object of the present invention is to provide particles for a diagnostic agent having excellent staining and dyeing properties for a dye or a fluorescent material.
Also, an object of the present invention is to provide particles for a diagnostic agent, which can be used suitably in a test for determining a substance to be tested, in which the substance to be tested capable of specifically binding to an antigen, an antibody or a nucleic acid is added to an aqueous dispersion of particles whose surfaces are sensitized with the antigen, antibody or nucleic acid to aggregate the particles, and changes in the turbidity caused by the aggregation of the particles are measured.
The present invention relates to a particle for a diagnostic agent, which comprises a polymer particle (hereinafter referred to as xe2x80x9cspecific (co)polymerxe2x80x9d) comprising
(A) from 20 to 100% by weight of a structural unit derived from at least one of acrylate having an aliphatic hydrocarbon group and methacrylate having an aliphatic hydrocarbon group (hereinafter referred to as xe2x80x9cstructural unit (A)xe2x80x9d),
(B) from 0 to 10% by weight of a structural unit derived from unsaturated carboxylic acid (hereinafter referred to as xe2x80x9cstructural unit (B)xe2x80x9d), and
(C) from 0 to 80% by weight of a structural unit derived from a vinyl monomer copolymerizable with the acrylate, methacrylate and unsaturated carboxylic acid (hereinafter referred to as xe2x80x9cstructural unit (C)xe2x80x9d).
Furthermore, the present invention relates to a process for producing the above particle for a diagnostic agent, which comprises
polymerizing in water a monomer comprising
(A) from 20 to 100% by weight of at least one member selected from the group consisting of acrylate having an aliphatic hydrocarbon group of 20 or less carbon atoms and methacrylate having an aliphatic hydrocarbon group of 20 or less carbon atoms,
(B) from 0 to 10% by weight of unsaturated carboxylic acid, and
(C) from 0 to 80% by weight of a vinyl monomer copolymerizable with the acrylate, methacrylate and unsaturated carboxylic acid to obtain a water dispersion of a polymer particle; and
alkalizing the water dispersion, followed by heating.
Moreover, the present invention relates to a turbidmetric iummunoassay which comprises
mixing a substance to be tested with the above particle for a diagnostic agent having thereon a biochemical substance capable of specifically reacting with the substance to be tested to aggregate the particle for a diagnostic agent, and
measuring an absorbance of the resulting mixture.